Thrombopoietin (TPO) protein, a cytokine, stimulates the creation and maturation of megakaryocytes. TPO is also referred to as thrombocytopoiesis-stimulating factor (TSF). TPO plays a major role in stimulating thrombocytopoiesis (platelet production). For a comprehensive review of TPO, see McDonald, The Amer. J. of Ped. Hem./Onc. 14 (1): 8-21 (1992), which is incorporated herein by reference. All references cited are incorporated hereinby reference.
Platelets are the blood cells responsible for clotting and low platelet counts can lead directly to excessive internal bleeding. Platelets--small blood cell fragments that break off from a relatively huge parent cell (the megakaryocyte)--are key to normal blood clotting. They routinely serve as patches for small defects in blood vessels to confine hemorrhage and encourage ordinary coagulation. Vast numbers of platelets aggregate to form a thrombus that stops excessive bleeding at wound sites.
In thrombocytopenia, blood platelet levels drop and cause abnormal bleeding into the skin, especially from small blood vessels. This is a serious and sometimes fatal condition. Thrombocytopenia is one of the most serious side effects of chemotherapy and irradiation. Thrombocytopenia also may result from infectious disease, leukemia, bone marrow transplants, certain diseases of the spleen, complications of pregnancy and child-bearing, gram-negative sepsis, complications of cardiac surgery, aplastic anemia and myelodysplastic syndromes.
With regards to chemotherapy, as many as 250,000 patients undergo such treatment each year in the United States alone, according to the FDA. Of those, 35% experience either severe or moderately severe thrombocytopenia. Last year, a total of about 10 million platelet transfusions were performed in the United States to enhance the blood's ability to clot.
Such platelet transfusions carry inherent risks of causing an immune response to the foreign platelets, transmitting viral diseases, such as HIV and hepatitis, and causing graft-vs-host disease and congestive heart failure.
TPO, by stimulating production of the body's own platelets, has the potential to restore platelet counts to near-normal levels in these patients. If platelets could be maintained at normal levels by administering TPO, it may be possible to provide patients with higher and potentially curative doses of standard cancer drugs without the limiting side effects.
Thrombocytopenia is also an important complication of infection with Human Immunodeficiency Virus Type-I (HIV-1). While HIV-related thrombocytopenia (HRT) may appear at any stage of HIV infection, the incidence of HRT appears to range from around 9% in HIV-infected, non-AIDS patients to around 21% in HIV infected patients with AIDS. Severity of thrombocytopenia has been correlated with the stage of the disease. See, Sloand, et al, Eur J Hematol, 48:168-172 (1992). HRT is a significant clinical problem, e.g., 40% of patients have platelet concentrations below 50,000/.mu.l and suffer bleeding episodes as a result, and 1 of 1004 patients in one study died as a result of intracranial hemorrhage. HRT is considered to be a Category B clinical condition in the CDC classification system for HIV infection. See, U.S. Dept. of Health, et al, Morbid Mortal Weekly Rep, (RR-17): 1-19 (1992).
The mechanism of thrombocytopenia in HIV infection has been the subject of considerable debate. It now seems clear that multiple mechanisms contribute to the clinical entity of HRT. HRT has been attributed to an autoimmune mechanism, supported by a common finding of elevated platelet-bound immunoglobulin G, and decreased platelet lifespan. See, Walsh, et al, N Eng J Med, 311:635-639 (1984); Ben-Ali, et al, Am J Hematol, 26:299-304 (1987). On the other hand, a defective production of platelets has also been proposed as a mechanism, supported by findings of morphological and quantitative abnormalities of megakaryocytes in the bone marrow of HIV- infected individuals. See, Zucker-Franklin, et al, Am J Pathol, 134:1295-1305 (1989); Scadden, et al, Blood, 1455-1463 (1989). Further, megakaryocytes internalize HIV-I viruses in the bone marrow. See, Zucker-Franklin, et al, Blood, 77:481-485 (1990). More recently, it has been suggested that the immune-mediated mechanism of platelet destruction is more important in early, asymptomatic HIV infection, whereas platelet-production defects predominate in later-stage patients with AIDS-related complex (ARC) or AIDS. See, Najean, et al, J Lab Clin Med, 123:415-420 (1994).
Murine leukemia virus infection in mice is well-recognized as a model for HIV-infection. See, Jolicoeur, FASEB J, 5:2398-2405 (1991); Morse, et al, AIDS, 6:607-621 (1992). This mouse model has been termed murine acquired immunodeficiency syndrome (MAIDS). The LP-BM5 murine leukemia virus (MuLV) is actually a mixture of ecotropic and mink cell focus forming MuLVs, which causes a lymphoproliferative disease in mice which is typified by lymphadenopathy, splenomegaly, and immunosuppression. See, Chow, et al, Exp. Hematol, 18:1038-1041 (1990). MAIDS has also been used as a model for HRT by examining the effects of azidothymidine (AZT) on platelet counts. In these studies, platelet counts typically fell to approximately 80% of control values, and were increased by therapy with AZT. See, Chow, et al, Exp. Hematol, 18:1038-1041 (1990); Chow, et al, J Lab Clin Med, 121:562-569 (1993); Chow, et al, Am J Hematol, 44:249-255(1993).
The use of AZT in humans, as has been widely reported, is not without significant risk of serious side effects. The risk of these side effects increases with increasingly higher dosages of AZT required to combat HIV/AIDS infections and raise platelet counts.
Particularly, then, there exists a serious and urgent need for a method and composition for treatment of HIV/AIDS-related thrombocytopenia, to raise platelet cell counts in such patents. Such method and composition would also advantageously reduce the need to use AZT to raise platelet counts in HIV/AIDS-infected patients.
More generally, there remains an urgent and unfulfilled need for a method and composition for the treatment of various thrombocytopenia-related conditions, such as result from the side effects of chemotherapy and irradiation in cancer patients, as well as other conditions described above, which would advantageously eliminate the need for risky platelet transfusions.
With respect to the structure of the human TPO molecule, TPO consists of 332 amino acid residues with a leader sequence of 21 amino acid residues. At residues 153 and 154, there is an Arg-Arg sequence, which is a degradation point. It has been reported that the first half of this molecule up to residue 153, which is the N-terminal region of the TPO molecule, has the same TPO activity as the whole molecule. This is based on the results that both the full-length and N-terminal fragment of TPO stimulated BaF.sub.3 /mpl cell in vitro. In this assay, supernatants from HEK 293 cells transfected with the sequence for the N-terminal domain had activity similar to that of supernatants from HEK 293 cells expressing the full-length TPO. See de Sauvage et al, Nature, 369:533-538 (1994).
The N-terminal fragment has been characterized as the erythropoietin-like (EPO-like) domain of TPO. Thus, it is disclosed in the de Sauvage publication that the EPO-like domain (N-terminal portion) is the mature or active moiety of TPO responsible for increasing platelet cell counts. In the de Sauvage publication, page 537, it states that the importance of the C-terminal region of the TPO molecule, which encompasses that portion from the Arg residue at amino acid position 154 to the end of the sequence, is unknown and remains to be elucidated. It is suggested that this region may act to stabilize and increase the half-life of circulating TPO. There is no suggestion in the de Sauvage publication that the C-terminal region or any portion of that region possesses any TPO activity.
As disclosed by de Sauvage, the C-terminal domain of TPO is not required for binding and activation of the c-Mpl receptor, which is the cytokine receptor for the active TPO moiety or ligand and which regulates megakaryocytopoiesis.
Further, it has been reported with regards to murine TPO that the junction between the N-terminal EPO-like domain and the C-terminal domain also contains a pair of arginine residues resembling a dibasic proteolytic site. It is disclosed that there is no evidence at this time to suggest that the recombinant c-Mpl ligand (TPO) produced in baby hamster kidney (BHK) cells is cleaved at this site to form the N- and C-terminal fragments. Lok et al, Nature, 369:565-568 (1994).
It would therefore certainly be a surprising and unexpected discovery if the C-terminal portion of the TPO molecule were found to be a moiety or ligand more active than the N-terminal domain for the therapeutic effect of advantageously increasing or raising platelet cell counts in thrombocytopenic patients.